Protein secretion is one of the most important issues of protein production in the field of biotechnology. This process is composed of the following steps: first, translocation across the endoplasmic reticulum (ER) membrane; second N-glycosylation and folding in the ER lumen; third, exit from the ER; fourth, modifications in the Golgi apparatus; and finally release from the secretory granules to the extracellular space (Sakaguchi 1997). Whether or not a protein is secreted from the cells mainly depends on whether it can be translocated across the membrane and whether it can be correctly folded in the ER lumen. Membrane translocation is obligatorily coupled in mammalian cells. After membrane translocation, the nascent peptides are released into the lumenal space and folded with the assistance of various chaperones and folding enzymes. Wrongly folded proteins are trapped within the ER and thus cannot proceed towards the secretory compartments. In biotechnological processes in which massive protein expression occurs, secretion can represent a bottleneck and limit the rate of expression.
Signal peptides or leader sequences, are located at the amino terminus of nascent polypeptides. They target proteins to the secretory pathway and are cleaved from the nascent chain once translocated in the reticulum endoplasmatic membrane.
The signal peptide consists of three regions: an amino-terminal polar region (N region), where positive charged aminoacid residues are frequently observed; a central hydrophobic region (H region) of more than 7-8 hydrophobic amino acid residues; and a carboxy-terminal region (C region) that includes the signal peptide cleavage site (Sakaguchi 1997). The eukaryotic H regions are dominated by Leu with some occurrence of Val, Ala, Phe and Ile. The cleavage of the signal peptide from the mature protein occurs at a specific site and the cleavage specificity resides in the last residue of the signal sequence (Nielsen et al. 1997). Close to the cleavage site −3 and −1 alanine is more predominant. This site confers processing specificity. No further specific patterns in the first few positions of the mature protein can be seen in eukaryotic organisms (Nielsen et al 1997). Therefore a “bad” signal peptide can promote more than one specific cleavage resulting in non-homogenous expression of the protein; i.e. the protein will be expressed with different N-terminal aminoacids.
Since many proteins are regulated under physiological conditions the use of natural regulatory signals for overexpression in mammalian systems is not desirable. For example, in such systems efficient promoters such as CMV and SV40 are used to control expression of recombinant proteins of interest. Similarly, the use of effective signal peptides such as SV40 and hGH poly A to overexpress recombinant secreted proteins instead of their endogenous counterparts would be advantageous.
The signal peptide of the human growth hormone (hGH) has been described to be effective in targeting the secretion of intracellular, membrane bound proteins and proteins secreted by different mechanisms than those governed by signal peptides.
For example, WO26562 describes the secretion of the intracellular protein icIL-1ra-II by fusion of the signal peptide of hGH to the sequence of the icIL-1ra-II. The invention relates to a process for the recombinant expression of a protein having the amino acid sequence of natural icIL-1ra-II in a recombinant cell expression system through use of a vector which is a fusion of the signal peptide of a human secretory protein, preferably the 26 amino acid signal peptide of hGH, fused in proper reading frame with the DNA encoding icIL-1ra-Il. The process comprises producing an expression vector containing DNA encoding icIL-1ra-II, either in the form of cDNA or genomic DNA, fused in proper reading frame with DNA encoding the selected signal peptide (SEQ ID NO:1), preferably the 26 amino acid hGH signal peptide (FIG. 1 SEQ ID NO:2). The expression vector is then inserted into an appropriate expression host, such as CHO cells. The transformed host cells are then cultured in a manner, which causes the expression vector to express its encoded protein, and the expressed and secreted icIL-1ra-II protein is then collected and purified from the culture medium.
Morris at al. (1999) describes the use of hGH signal peptide for the secretion of the protein CDL40L, which exists in nature predominantly as a membrane-anchored molecule. Several reports have shown that the soluble form of CD40L is biologically active (Fanslow et al. 1994, Hollenbaugh et al. 1992 and Mazzei et al. 1995). To use CD40L as a potential therapeutic, optimisation of soluble forms of this molecule have been developed. In this work, the activity of soluble forms of CD40L, and the activity of the soluble multimerized CD40L TNF homologous region, have been compared. The soluble forms of CD40L have been prepared by fusion of the entire extracellular domain of human CD40L or the CD40L region homologous to TNF sequence to the signal peptide sequence of hGH. The multimerized form of the CD40L has been prepared by fusion to an isoleucine zipper (IZ). The results showed that multimerization increases the activity of soluble CD40L.
Pecceu et al (1991) describes the use of the hGH signal peptide to express and secrete the mature form of IL-1β. In the body, after synthesis, proIL-1β remains primarily cytosolic until it is cleaved and transported out of the cells. Examination of the sequence reveals the absence of a classical N-terminal or internal hydrophobic signal peptide. Release of mature IL-1β appears to be linked to processing at the aspartic acid-alanine peptide cleavage by the converting enzyme (ICE) (Dinarello 1996). Although ICE is constitutively expressed in most cells, not all cells process proIL-1β and secrete mature IL-1β. Therefore secretion of mature IL-1β is cell dependent. Pecceu discloses the use a recombinant vector containing only the DNA encoding the mature form of IL-1β, without any signal peptide and a vector containing the DNA encoding the mature form of IL-1β joined to hGH signal peptide. The results show that only 52% of the protein are secreted using the first construct, while using the construct with hGH signal peptide results in 97% secretion.
The first ATG codon for initiation of translation has to be identified by the transcriptional machinery. An ATG codon in a very weak context is not likely to be the start site for translation. The optimal context for initiation of translation in vertebral mRNAs is a G residue following the ATG codon (position +4 in the coding region) and a purine, preferable A, three nucleotides upstream (−3 in the noncoding region) this consensus sequence has been designated Kozak sequence (Kozak 1996, 1999). Messenger RNA in which the first ATG codon lacks the preferred nucleotides in both of these key flanking positions (a “bad” or non optimal Kozak sequence) have the special property of initiating translation at the first and second ATG codons, thereby producing two proteins from one RNA. The ATG in the initiation site of hGH signal peptide is followed by G (in position +3) required for obtaining an optimal Kozak sequence (FIGS. 1 and 2 SEQ ID NO:3), which ensures the start of translation at the first ATG site only and homogeneity of the product.
IL-18 binding protein (IL18-BP) was affinity purified from human urine using IL-18, sequenced and cloned. IL-18BP was found to abolish in vitro the activity of the pro-inflammatory cytokine IL-18. (Novick et al. 1999). The DNA encodes a signal peptide at its N-terminal portion. Part of the Kozak sequence encoded inside the signal peptide is not of the appropriate context.
IL-18BP leaderATGA+1+4ATGGKozak Concensus+1+4Human growth hormone leader+1+4
Many naturally occurring proteins and enzymes are multimeric. Examples include hemoglobin, antibody, thyrotropin (TSH), fertility hormones such as follicle stimulating hormone (FSH), luteinizing hormone (LH) and human choriogonadotropin (HCG). The subunits of a multimeric protein may be identical, homologous or totally dissimilar and dedicated to different tasks.
Follicle stimulating hormone is known to be useful in the treatment of infertility. FSH is comprised of two polypeptide subunits, alpha and beta. Preparations containing this hormone have been employed to assist in effecting pregnancy using both in-vivo and in-vitro techniques. Human FSH has been isolated from human pituitary glands and from post-menopausal urine. More recently, it has been produced using recombinant DNA techniques (EP0211894B).
Mountford et al. described the purification and characterization of ovine FSH secreted by CHO cell lines stably transfected with cDNA constructs encoding the alpha and beta sub-units of this hormone. Replacement of the 5′ untranslated and signal peptide-coding sequence with those from an ovine growth hormone resulted in a mixed population of beta sub-unit polypeptides derived from two cleavage sites.
Thus, new methods for improving homogeneity of production of recombinant proteins comprising more than one polypeptide subunit are needed.
It has been found in accordance with the present invention that mammalian engineered cells harbouring DNA encoding human FSH, and in the DNA the endogeneous human FSH signal peptide was replaced with the human growth hormone signal peptide, produced more homogenous FSH.